SUMMARY Melanoma is a skin cancer that accounts for only 4% of cases but 80% of deaths; in 2014 melanoma resulted in ~10,000 deaths in the United States. Spontaneous development of CD8+ T cell immunity against melanoma is associated with improved clinical outcome (1, 2), indicating the potential for immunotherapy to increase patient survival. However, the protective effect of CD8+ T cells reactive against even highly immunogenic melanoma antigens is often diminished (3-7), despite the presence of functional T cells in the systemic circulation. The precise molecular nature of this tumor-infiltrating lymphocyte (TIL) dysfunction is not well understood, underscoring the need for new approaches, including physical science methods to study the unique pathophysiology of immune suppression in melanoma. We (8) and others (9-15) have shown that CD8+ T cell immunity, including melanoma rejection (8), depends on the affinity and durability of T cell receptor (TCR) and CD8 coreceptor binding to peptides bound to major histocompatibility complex (pMHC) (16, 17). How the tumor microenvironment (TME) impacts the mechanisms of molecular recognition to manifest deficient anti-melanoma immunity remains largely undefined. We recently discovered that the quantity (affinity) and quality (durability) of TCR and/or CD8 bonds with pMHC under force on TILs from in vivo primary murine melanomas are significantly reduced relative to T cells from the spleen and blood. Our findings are provocative since, despite striking differences between bond quantities and qualities, all analyzed cells display identical TCRs and were specifically linked to reduced CD8+ T cell effector functions. We hypothesize that deficient CD8+ T cell immunity in melanoma results at least in part from impaired antigen recognition within the TME, as manifested by reduced quantity and quality of TCR bonds with pMHC. The overall objective of the parent grant is to identify the mechanisms by which the TME suppresses TCR mechanosensing through the implementation of innovative in situ measurement techniques, advanced preclinical in vivo models of melanoma, and human clinical samples. The three specific aims of the parent grant are designed to answer the following three crucial scientific questions in melanoma mechanoimmunology: What molecular interactions crucial to T cell antigen recognition are impaired by the TME? What are the functional consequences of suppressed T cell antigen recognition? What are the mechanisms underlying the TME suppression of T cell antigen recognition?